Gene editing in zebrafish: a Hampden-Sydney student summer research experience at UAB

By Luke Bloodworth ’18

This summer I have been working with Dr. Anil Challa at the University of Alabama-Birmingham (UAB) with CRISPR, a new technology for the editing and knockout of genes.  The research we are doing at UAB with genetic engineering, given some training, would be something I would hope to be able to keep doing in Hampden-Sydney’s biology department. Dr. Challa has also expressed interest in helping us set up a zebrafish work bench so we could do our own exploration and research at HSC (He and I will present a joint seminar at H-SC in late January 2017).

The author injecting RNA samples into a polyacrylamide gel

The author injecting RNA samples into a polyacrylamide gel

Injecting fresh Zebrafish Embryos with CRISPR Cas-9 to knockout selected genes

Injecting fresh Zebrafish Embryos with CRISPR Cas-9 to knockout selected genes

So far I have used a cloud based biotech program called Benchling to locate specific spots on the amino acid coding exons of the genes we are knocking out. Taking this information, we used ensemble and CRISPR scanner to cross reference and further determine, hopefully, the proper and best binding sites for the CRISPR. Using this data, we have sequenced the proper sgRNAs (oligos) and created correlating primers for those CRISPRs. The next step will be embryo injections followed by genotyping and phenotyping.

Checking over oligonucleotides for CRISPR gene knockout in zebrafish

Checking over oligonucleotides for CRISPR gene knockout in zebrafish

I am also hoping to do a project on Argonaute (Ago). Ago is a DNA-guided DNA endonuclease, requiring no PAM sequence, and using a 24-nucleotide ssDNA with its 5′ end phosphorylated.

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